PROJECT 2: PROJECT SUMMARY With the rise in incidence of metastatic melanoma and prolonged survival of patients with this disease, an ever increasing number of patients are living with metastatic melanoma and requiring therapy. Not all patients respond to PD-1 inhibitors, and there is therefore an urgent need for means to predict response to this class of agents, to monitor patients for response, and to understand mechanisms of sensitivity and resistance to these drugs that might be harnessed for designing effective combination regimens. Although tumor cell properties, such as PD- L1 expression, are somewhat associated with response, mounting evidence generated suggests that properties and quantity of tumor infiltrating inflammatory cells might be more informative in terms of predicting and monitoring response and can enable us to determine approaches to overcome resistance. However, melanomas often metastasize to areas that are difficult to biopsy, such as the brain, spleen, small bowel and bone, and we therefore propose approaches to studying properties of tumor infiltrating lymphocytes (TIL) by isolating and profiling T cells that originated in the tumor but can be identified in the circulation. In previous studies we demonstrated that these circulating T cells, which express both PD-1 and TIM-3, are specific to the tumor microenvironment, and are not found in the circulation of healthy individuals or patients with autoimmune disease. We propose to conduct single cell analysis of these TIM3+PD-1+ circulating CD4 and CD8 cells that have been in tumor tissue using state-of-the-art technologies. We have assembled a team of interdisciplinary researchers with the goal of developing liquid biopsy methods to determine mechanisms of resistance to immune checkpoint inhibitors. We will start by conducting single cell CyTOF and functional cytokine analysis of CD4 and CD8 cells from the circulation and tumor tissue to determine similarities and differences at the molecular level. We will then barcode TIM3+PD-1+ cells using TcR chain sequences to identify circulating TIL by the presence of TcR sequences that are identical to TIL isolated from tumors representing sister clones. Using single cell RNA- seq, we will therefore be able to assess TIL function by studying function in this population of circulating TIL. This will be done on prospectively collected matched samples from melanoma patients undergoing resection of metastases, including brain metastases, biopsies from other sites and blood samples. Our liquid biopsy technique then be studied on blood samples from patients treated with PD-1 inhibitors before and on therapy, with complete clinical annotation including response to therapy. If successful, our liquid biopsy approach can be applied to other treatment settings and other tumor types, and thus has the potential to significantly impact patient care by enabling improved patient selection and avoiding the morbidity and expense associated with repeat tumor biopsies. Moreover, the single cell analysis will enable us to better understand mechanisms of resistance, which in turn can facilitate drug development for patients with tumors resistant to PD-1 inhibitors.